1. Field of the Invention
The present invention relates to a method and an apparatus for the quantitative determination of micro-organisms or the like. More particularly, it relates to a method and an apparatus for the quantitative determination of micro-organisms or pyrogens in a liquor, which are simple and trustworthy.
2. Description of the Prior Art
Determination (quantitative determination) of the amount of micro-organisms in various media is important for public sanitation or quality control in, not only the fields of positively utilizing micro-organisms, such as fermentation, brewing, etc., but also various other fields such as medical treatment, food stuffs, analysis, etc.
As methods for the quantitative determination of such micro-organisms, the following methods have been generally known, hitherto;
1 Visual estimation method (so-called colony method); a method for the quantitative determination of micro-organisms wherein a test liquor containing micro-organisms is, after subjecting to a dyeing treatment, put on a slide glass, the number of micro-organisms in the visual field is counted by a microscope, and the amount of the micro-organisms is determined on the basis of said number.
2 Optical estimation method (so-called OD method); a method for the quantitative determination of micro-organisms wherein a test liquor containing micro-organisms is subjected to irradiation of light to find out its turbidity and the amount of the micro-organisms is determined on the basis of said turbidity.
3 Dilution culture method; a method for the quantitative determination of micro-organisms wherein a prescribed dilution series of a sample liquor containing micro-organisms is prepared, each diluted liquor is inoculated and cultivated in a culture tube, and the amount of the micro-organisms is determined statistically on the basis of the state of their growing.
On the other hand, pyrogens are known as substances which cause difficulties in various media just like micro-organisms, though they are not micro-organisms, and quantitative determination of such pyrogens is also desired for public sanitation or quality control. Especially, quantitative determination of pyrogens in distilled water, such as distilled water for injection, distilled water for dialysis, etc., is important for public sanitation.
As methods for quantitative determination or test methods of such pyrogens, a method relying on the elevation of body temperature of rabbits that is caused by injection to the rabbits of a subject sample (the Japanese Pharmacopoeia: test methods for pyrogens, is referred to) is widely used. However, this method involves inconveniences that the results are often influenced by the individual difference in sensibility among the rabbits and, moreover, observation of the changes in body temperature and breeding of the animals are required over a long time period. Accordingly, the so-called Limulus test is recently remarked as a method for the quantitative determination and evaluation of pyrogens, wherein the selective reaction between a blood-corpuscle extract of Limulus and a pyrogen is utilized and the amount of the pyrogen is determined on the basis of the gelation degree by said reaction or the absorbance after diazotization of p-nitroaniline which may produce from said reaction performed in the presence of a color-forming synthetic substrate (refer to a Japanese Journal "Kan-Tan-Sui" 12(4): 523-528, 1986, etc.).
However, the hitherto known methods for the quantitative determination of micro-organisms as mentioned above involve various problems that the operation for determination requires a long time and, as well, is troublesome and requests skillfulness, that the range of the amount of micro-organisms capable of quantitative determination is narrow and the results of determination are not necessarily trustworthy, and also that they are not suitable for continuous determination.
On the other hand, the above-mentioned, hitherto used methods for quantitative determination and evaluation of pyrogens also involve problems that the operation requires a long time and, as well, is troublesome and requests skillfulness, that they use a costly Limulus extract, and also that they are not suitable for continuous determination.
The present invention has been accomplished in view of such circumstances with the purpose of providing a method and an apparatus for quantitative determination of the amount of micro-organisms or pyrogens, which are simple and trustworthy and, as well, suitable for continuous determination.